Lactoferrin in the reduction of circulating cholesterol, vascular inflammation, atherosclerosis and cardiovascular disease

ABSTRACT

The present invention relates to methods of using lactoferrin (LF) to reduce circulating levels of cholesterol and vascular inflammation, in order to treat, prevent or reduce the incidence of atherosclerosis and cardiovascular disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 10/728,275filed Dec. 4, 2003 which claims the benefit of U.S. ProvisionalApplications 60/498,337 filed Aug. 27, 2003 and 60/430,867 filed Dec. 4,2002, both of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to methods of using lactoferrin (LF) toreduce circulating levels of cholesterol and vascular inflammation, inorder to treat, prevent or reduce the incidence of atherosclerosis andcardiovascular disease. More particularly, the present invention relatesto methods of reducing circulating levels of cholesterol and vascularinflammation by administering a composition of lactoferrin.

BACKGROUND OF THE INVENTION

According to current estimates, 61,800,000 people in America have one ormore forms of cardiovascular disease. These diseases claimed 958,775lives in 1999 (40.1 percent of all deaths). Atherosclerosis is a leadingform of cardiovascular disease, which involves the slow build-up offatty plaques on the arterial wall. This build-up can damage thevascular endothelium causing inflammation, a narrowing of the arteriesand potential arterial blockages that can result in heart attacks.Atherosclerosis is a complex disease that starts in childhood and oftenprogresses when people grow older. In some people it progresses rapidly,even in their third decade. Elevated levels of cholesterol, inparticular LDL (low-density lipoprotein), and triglycerides in the bloodhave been associated with the development of fatty plaques, which canlead to generalized vascular damage, atherosclerosis and eventuallyheart attack. Atherosclerosis and cardiac disease is also associatedwith increased cardiovascular inflammation, specifically as measured bylevels of circulating C-reactive protein (CRP).

One key strategy for reducing the risk of atherosclerosis has been tolower the levels of cholesterol in the blood. Cholesterol levels in manypeople can be controlled by diet, but for many patients diet changesalone are insufficient to reduce high cholesterol. In recent years,cholesterol lowering drugs such as Zocor® (simvastatin) and Lipitor®(atorvastatin) have been increasingly prescribed to help patients lowertheir cholesterol levels. These drugs however, are not equally effectivein all patients and frequently are associated with significant adverseside effects. A second key emerging strategy is the reduction of CRP, animportant indicator of vascular inflammation and independentlyassociated with increased risk of cardiovascular disease. Thus, saferand more effective treatments for lowering cholesterol and for reducingthe vascular inflammation associated with atherosclerosis are of greatpotential value.

Lactoferrin is a single chain metal binding glycoprotein. Many celltypes, such as monocytes, macrophages, lymphocytes, and brush-bordercells in the intestine, are known to have lactoferrin receptors.Lactoferrin is found mainly in external secretions of mucosal epitheliasuch as breast milk, saliva, tears, bile, and pancreatic fluid and has awide array of functions related to host primary defense mechanisms. Forexample, lactoferrin has been reported to activate natural killer (NK)cells, induce colony stimulating activity, activate polymorphonuclearneutrophils (PMN), regulate granulopoeisis, enhance antibody-dependentcell cytotoxicity, stimulate lymphokine-activated killer (LAK) cellactivity, and potentiate macrophage toxicity.

Recombinant human lactoferrin has previously been described as beingpurified after expression in a variety of prokaryotic and eukaryoticorganisms including aspergillus (U.S. Pat. No. 6,080,559), cattle (U.S.Pat. No. 5,919,913), rice, corn, Sacharomcyes (U.S. Pat. No. 6,228,614)and Pichia pastoris (U.S. Pat. No. 6,455,687, 6,277,817, 6,066,469).Also described are expression systems for the expression of full-lengthhuman lactoferrins (e.g., U.S. Pat. No. 6,100,054). In all cases, partof the teaching is expression of the full length cDNA and purificationof the intact protein whose N-terminal, after processing of the leaderpeptide, is the amino acid glycine. Nuijens et al. (U.S. Pat. No.6,333,311) separately describe variants of human lactoferrin but theirfocus is limited to deletion or substitution of arginine residues foundin the N-terminal domain of lactoferrin.

The present invention is the first to use a lactoferrin composition as ameans of reducing cholesterol and cardiovascular inflammation and fortreating or reducing atherosclerosis and cardiovascular disease.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a method for modulating circulatinglevels of cholesterol and reducing the vascular inflammation associatedwith atherosclerosis and cardiovascular disease. The method of treatmentinvolves administration of a lactoferrin composition.

The lactoferrin composition, which is dispersed in a pharmaceuticallyacceptable carrier, comprises lactoferrin or an N-terminal lactoferrinvariant in which at least the N-terminal glycine residue is truncated orsubstituted. The lactoferrin is mammalian lactoferrin, moreparticularly, the lactoferrin is human or bovine. Yet further, thelactoferrin is recombinant lactoferrin. N-terminal lactoferrin variantsinclude variants that at least lack the N-terminal glycine residue orcontain a substitution at the N-terminal glycine residue. Thesubstitution can comprise substituting a natural or artificial aminoacid residue for the N-terminal glycine residue. For example, thesubstitution can comprise substituting a positive amino acid residue ora negative amino acid residue for the N-terminal glycine residue orsubstituting a neutral amino acid residue other than glycine for theN-terminal glycine residue. Other N-terminal lactoferrin variantsinclude lactoferrin lacking one or more N-terminal residues or havingone or more substitutions in the N-terminal. In specific embodiments,the N-terminal lactoferrin variant comprises at least 1% of thelactoferrin composition, at least 5% of the lactoferrin composition, atleast 10% of the lactoferrin composition, at least 25% of thelactoferrin composition, at least 50% of the lactoferrin composition orany range in between.

The amount of the lactoferrin that is administered is about 1 ng toabout 20 g per day, more preferably, the amount is about 0.1 g to about5 g per day. More particularly, the composition is a solution, capsuleor a tablet having a lactoferrin concentration of about 0. 1% to about100%.

In further embodiments, a metal chelator dispersed in a pharmaceuticallyacceptable carrier can also be administered with the lactoferrincomposition. Preferred metal chelator include, but are not limited toethylenediaminetetraacetic acid (EDTA) or[ethylenebis(oxyethylenenitrilo)] tetraacetic acid (EGTA). Morepreferably, the metal chelator is EDTA. The amount of EDTA that isadministered is about 1 ng to about 1 g per day.

An embodiment of the present invention is a method of treating acardiovascular disease comprising the step of administering to a subjecta lactoferrin composition in an effective amount to provide animprovement in the cardiovascular disease, for example, atherosclerosis.The lactoferrin composition reduces the levels of circulating totalcholesterol, low density lipoproteins (LDL), or very low densitylipoproteins (VLDL). Still further, the lactoferrin compositionincreases the levels of circulating high density lipoproteins (HDL). Inaddition to modulating levels of cholesterol, the lactoferrincomposition reduces the levels of vascular inflammation, circulatingC-reactive protein (CRP), triglycerides, or proliferation of vascularsmooth muscle cells. The lactoferrin composition may also reducevascular spasms or vascular hyper-reactivity or promote endothelialintegrity or healing. In further embodiments, the lactoferrincomposition reduces the production or activity of pro-inflammatorycytokines.

The lactoferrin composition of the present invention can be administeredparenterally, for example, subcutaneously, intramuscularly,intraperitoneally, intravenously, intraarterially, intramyocardially,transendocardially, transepicardially, or intrathecally.

In a further embodiment, the lactoferrin composition is administeredorally. For oral administration, an antacid in combination with thelactoferrin composition can be administered. The lactoferrin can beformulated in a delayed release formulation. Still further, thelactoferrin composition can be formulated wherein release occurs in thesmall intestine or in the large intestine.

Another embodiment of the present invention is a method of modulatingatherosclerosis in a subject comprising the step of administering to thesubject a lactoferrin composition in an effective amount to modulateatherosclerosis in the subject. Modulating is reducing the incidence ofatherosclerosis or reducing the severity of atherosclerosis. In furtherembodiments, the lactoferrin composition can be administered incombination with an anti-cholesterol agent or an anti-inflammatoryagent. The anti-cholesterol agent is selected from the group consistingof cholesterol absorption inhibitors, bile acid sequestrants(cholestryramine, cholestipol and colesevalam), nicotinic acid, fibricacids (gemfibrozil, fenofibrate and clofibrate) and HMG-coA reductaseinhibitors (lovastatin, pravastatin, simvastatin, fluvastatin,atorvastatin and cerivastatin).

Another embodiment is a method of preventing a cardiovascular disease ina subject at risk for developing a cardiovascular disease comprising thestep of administering to the subject a lactoferrin composition in anamount sufficient to result in prophylaxis of the cardiovascular diseasein the subject. The cardiovascular disease is atherosclerosis.

Still further, another embodiment is a method of reducing the risk ofcardiovascular disease in a subject at risk for developing acardiovascular disease comprising the step of administering to thesubject a lactoferrin composition in an effective amount to result in areduction of risk of the cardiovascular disease in the subject. Thecardiovascular disease is atherosclerosis.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated that the conception and specific embodimentdisclosed may be readily utilized as a basis for modifying or designingother structures for carrying out the same purposes of the presentinvention. It should also be realized that such equivalent constructionsdo not depart from the invention as set forth in the appended claims.The novel features which are believed to be characteristic of theinvention, both as to its organization and method of operation, togetherwith further objects and advantages will be better understood from thefollowing description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawings.

FIG. 1 shows the net reduction of total serum cholesterol in subjectsreceiving oral rhLF for seven days with respect to patients receivingplacebo for seven days.

FIG. 2 shows the reduction of C-reactive protein in subjects receivingoral rhLF for seven days.

FIG. 3A and FIG. 3B show the effect of rhLF on hyperlipidemia in mice.FIG. 3A shows the effect of rhLF on HDL, LDL and total cholesterollevels. FIG. 3B shows the effect on HDL/LDL ratio.

FIG. 4 shows a dose effect of rhLF on hyperlipidemia in mice.

FIG. 5 shows the effect of oral rhLF with lovastatin on hyperlipidemiain mice.

DETAILED DESCRIPTION OF THE INVENTION

It is readily apparent to one skilled in the art that variousembodiments and modifications can be made to the invention disclosed inthis Application without departing from the scope and spirit of theinvention.

As used herein, the use of the word “a” or “an” when used in conjunctionwith the term “comprising” in the claims and/or the specification maymean “one,” but it is also consistent with the meaning of “one or more,”“at least one,” and “one or more than one.” Still further, the terms“having”, “including”, “containing” and “comprising” are interchangeableand one of skill in the art is cognizant that these terms are open endedterms.

The term “atherosclerosis” as used herein includes a form ofarteriosclerosis characterized by a combination of changes in the intimaof arteries, such changes include, but are not limited to accumulationof lipids, complex carbohydrates, blood and blood products, fibroustissue and calcium deposits. Yet further, atherosclerotic plaques can becharacterized into at least two areas. One type is characterized byprominent proliferation of cells with small accumulations of lipids. Thesecond type consists mostly of intracellular and extracellular lipidaccumulation and a small amount of cellular proliferation.

The term “cardiovascular disease or disorder” as used herein refers todisease and disorders related to the cardiovascular or circulatorysystem. Cardiovascular disease and/or disorders include, but are notlimited to, diseases and/or disorders of the pericardium, heart valves(e.g., incompetent valves, stenosed valves, rheumatic heart disease,mitral valve prolapse, aortic regurgitation), myocardium (e.g., coronaryartery disease, myocardial infarction, heart failure, ischemic heartdisease, angina) blood vessels (e.g., hypertension, arteriosclerosis,aneurysm) or veins (e.g., varicose veins, hemorrhoids). Yet further, oneskill in the art recognizes that cardiovascular diseases and/ordisorders can result from congenital defects, genetic defects,environmental influences (e.g., dietary influences, lifestyle, stress,etc.), and other defects or influences.

The term “chemokine” as used herein refers to small cytokines that areinvolved in the migration and activation of cells, for examplephagocytic cells and lymphocytes. One of skill in the art realizes thatchemokines play a central role in inflammatory and immune responseprocesses.

The term “cholesterol” as used herein refers to the monohydric alcoholform, which is a white, powdery substance that is found in all animalcells and in animal-based foods (not in plants). Cholesterol is anessential nutrient necessary for many functions, including thefollowing: repairing cell membranes, manufacturing vitamin D on theskin's surface, production of hormones, such as estrogen andtestosterone, and possibly helping cell connections in the brain thatare important for learning and memory.

The term “chylomicron” as used herein refers to the largest in size andlowest in density of the triglyceride carrying lipoproteins.

The term “cytokine” as used herein refers to proteins that are made bycells that affect the behavior of other cells, for example stimulate orinhibit cell proliferation. For example, cytokines that are made bylymphocytes are often called lymphokines or interleukins. One of skillin the art realizes that the term cytokine is a generic term used in theliterature to refer to proteins that are made by cells that can effectthe behavior of other cells.

The term “effective amount” or “therapeutically effective amount” areinterchangeable as used herein and refer to an amount that results in animprovement or remediation of the symptoms of the disease or condition.

The term “high-density lipoprotein” or “HDL” as used herein is thesmallest and most dense type of cholesterol-carrying lipoprotein and isoften referred to as the “good” cholesterol.

The term “intermediate density lipoprotein” or “IDL” as used hereinrefers to a triglyceride-carrying lipoprotein.

The term “lactoferrin” or “LF” as used herein refers to native orrecombinant lactoferrin. Native lactoferrin can be obtained bypurification from mammalian milk or colostrum or from other naturalsources. Recombinant lactoferrin (rLF) can be made by recombinantexpression or direct production in genetically altered animals, plants,fungi, bacteria, or other prokaryotic or eukaryotic species, or throughchemical synthesis.

The term “lactoferrin composition” as used herein refers to acomposition having lactoferrin, a portion or part of lactoferrin, anN-terminal lactoferrin variant, or a combination thereof.

The term “lipid” as used herein refers to the building blocks of any ofthe fats or fatty substances found in animals and plants, which arecharacterized by their insolubility in water and solubility in fatsolvents such as alcohol, ether and chloroform. Lipids include fats(e.g., esters of fatty acids and glycerol); lipoids (e.g.,phospholipids, cerebrosides, waxes) and sterols (e.g., cholesterol).

The term “lipoproteins” as used herein are protein spheres thattransport cholesterol, triglyceride, or other lipid molecules throughthe bloodstream. Lipoproteins are categorized into five types accordingto size and density. They can be further defined by whether they carrycholesterol [the two smaller lipoproteins (HDL and LDL)] ortriglycerides [the three largest lipoproteins (IDL, VLDL, andchylomicrons)].

The term “low density lipoprotein” or “LDL” as used herein is a type ofcholesterol-carrying lipoprotein which is often called the “bad”cholesterol.

The term “N-terminal lactoferrin variant” as used herein refers tolactoferrin wherein at least the N-terminal glycine has been truncatedand/or substituted. N-terminal lactoferrin variants also include, butare not limited to deletion and/or substitution of one or moreN-terminal amino acid residues, for example 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, or 16 N-terminal amino acid residues, etc. Thus,N-terminal lactoferrin variants comprise at least deletions ortruncations and/or substitutions of 1 to 16 N-terminal amino acidresidues. The deletion and/or substitution of at least the N-terminalglycine of lactoferrin mediates the same biological effects asfull-length lactoferrin and/or may enhance lactoferrin's biologicalactivity, for example by stimulating the production of various cytokines(e.g., IL-18, MIP-3α, GM-CSF or IFN-γ) by inhibiting various cytokines,(e.g., IL-2, IL-4, IL-5, IL-10, or TNF-α) by improving a cardiovasculardisease, e.g., atherosclerosis, or the parameters relating tocardiovascular disease including circulating levels of totalcholesterol, HDL, LDL, VLDL, trigylcerides and C-reactive protein (CRP).

The term “metal chelator” as used herein refers to a compound whichbinds metal. Metal chelators that can be used in the present inventioninclude the divalent metal chelators, for example,ethylenediaminetetraacetic acid (EDTA), [ethylenebis(oxyethylenenitrilo)] tetraacetic acid (EGTA),1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA),hydroxyethlene triamine diacetic acid, (HEDTA) or salts thereof.

The term “oral administration” as used herein includes oral, buccal,enteral or intragastic administration.

The term “parenteral administration” as used herein includes any form ofadministration in which the compound is absorbed into the subjectwithout involving absorption via the intestines. Exemplary parenteraladministrations that are used in the present invention include, but arenot limited to intramuscular, intravenous, intraperitoneal, intraocular,subcutaneous or intraarticular administration. Yet further, parenteraladministration also includes administration into a surgical field.

The term “pharmaceutically acceptable carrier” as used herein includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like.The use of such media and agents for pharmaceutically active substancesis well known in the art. Except insofar as any conventional media oragent is incompatible with the vectors or cells of the presentinvention, its use in therapeutic compositions is contemplated.Supplementary active ingredients also can be incorporated into thecompositions.

The term “preventing” as used herein refers to minimizing, reducing orsuppressing the risk of developing a disease state or parametersrelating to the disease state or progression or other abnormal ordeleterious conditions.

The term “statin” as used herein includes compounds that are HMG-CoAreductase inhibitors, for example, but not limited to simvastatin(Zocor®) and atorvastatin (Lipitor®). Thus, as used herein the terms“statin” and “HMG-CoA reductase inhibitor” are interchangeable.

The term “subject” as used herein, is taken to mean any mammaliansubject to which a lactoferrin composition is administered according tothe methods described herein. Thus, a skilled artisan realizes that amammalian subject, includes, but is not limited to humans, monkeys,horses, pigs, cows, dogs, cats, rats and mice. In a specific preferredembodiment, the methods of the present invention are employed to treat ahuman subject. In more preferred embodiments, the subject has signs orindicators of atherosclerosis. These signs or indicators include, forexample, the development of cholesterol plaques in the arteries andcalcification, the extent of which can be determined by Sudan IVstaining, or the development of foam cells in an artery or arterialspasm. Atherosclerosis also is characterized by a narrowing of thearteries detected by, for example, coronary angioplasty, ultrasound andultrafast CT. In further embodiments, the subject is at risk ofdeveloping a cardiovascular disease. Thus, the subject may or may not becognizant of their disease state or potential disease state and may ormay not be aware that they are need of treatment (therapeutic treatmentor prophylactic treatment).

The term “topical administration” as used herein includes, but is notlimited to topical, dermal, or epidermal.

The term “total cholesterol” as used herein refers to the sum of threekinds of lipids: high-density lipoprotein (HDL), low-density lipoprotein(LDL), and triglycerides. Levels of serum total cholesterol of >200mg/dl are levels that are an indicating risk factor for atherosclerosisand cardiovascular disease.

The term “triglycerides” as used herein are composed of fatty acidmolecules and are the basic chemicals contained in fats in both animalsand plants.

The term “treating” and “treatment” as used herein refers toadministering to a subject a therapeutically effective amount of arecombinant human lactoferrin composition so that the subject has animprovement in a cardiovascular disease or the parameters relating tocardiovascular disease including circulating levels of totalcholesterol, HDL, LDL, VLDL, trigylcerides and C-reactive protein (CRP).The improvement is any observable or measurable improvement. Thus, oneof skill in the art realizes that a treatment may improve the patientcondition, but may not be a complete cure of the disease.

The term “very low density lipoprotein” or “VLDL” as used herein refersto a triglyceride carrying lipoprotein.

A. LACTOFERRIN

The lactoferrin used according to the present invention can be obtainedthrough isolation and purification from natural sources, for example,but not limited to mammalian milk. The lactoferrin is preferablymammalian lactoferrin, such as bovine or human lactoferrin. In preferredembodiments, the lactoferrin is produced recombinantly using geneticengineering techniques well known and used in the art, such asrecombinant expression or direct production in genetically alteredanimals, plants or eukaryotes, or chemical synthesis. See, for example,U.S. Pat. Nos. 5,571,896; 5,571,697 and 5,571,691, which are hereinincorporated by reference.

In certain aspects, the present invention provides lactoferrin variantshaving enhanced biological activities over natural LF and or rLF, e.g.,the ability to stimulate and/or inhibit cytokines or chemokines. Inparticular, the invention provides variants of lactoferrin from which atleast the N-terminal glycine residue has been substituted and/ortruncated. The N-terminal lactoferrin variants may occur naturally ormay be modified by the substitution or deletion of one or more aminoacids.

The deletional variants can be produced by proteolysis of lactoferrinand/or expression of a polynucleotide encoding a truncated lactoferrinas described in U.S. Pat. No. 6,333,311, which is incorporated herein byreference.

Substitutional variants or replacement variants typically contain theexchange of one amino acid for another at one or more sites within theprotein. Substitutions can be conservative, that is, one amino acid isreplaced with one of similar shape and charge. Conservativesubstitutions are well known in the art and include, for example, thechanges of: alanine to serine; arginine to lysine; asparagine toglutamine or histidine; aspartate to glutamate; cysteine to serine;glutamine to asparagine; glutamate to aspartate; glycine to proline;histidine to asparagine or glutamine; isoleucine to leucine or valine;leucine to valine or isoleucine; lysine to arginine; methionine toleucine or isoleucine; phenylalanine to tyrosine, leucine or methionine;serine to threonine; threonine to serine; tryptophan to tyrosine;tyrosine to tryptophan or phenylalanine; and valine to isoleucine orleucine.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982). It is accepted thatthe relative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte and Doolittle,1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, e.g., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those that are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine(−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5);tryptophan (−3.4).

Still further, it is understood that an amino acid can be substitutedfor another having a similar hydrophilicity value and still obtains abiologically equivalent and immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those that are within ±1 are particularlypreferred, and those within ±0.5 are even more particularly preferred.

Thus, in the present invention, substitutional variants or replacementcan be produced using standard mutagenesis techniques, for example,site-directed mutagenesis as disclosed in U.S. Pat. Nos. 5,220,007;5,284,760; 5,354,670; 5,366,878; 5,389,514; 5,635,377; 5,789,166, and6,333,311, which are incorporated herein by reference. It is envisionedthat at least the N-terminal glycine amino acid residue can be replacedor substituted with any of the twenty natural occurring amino acids, forexample a positively charged amino acid (arginine, lysine, orhistidine), a neutral amino acid (alanine, asparagine, cysteine,glutamine, glycine, isoleucine, leucine, methionine, phenylaline,proline, serine, threonine, tryptophan, tyrosine, valine) and/or anegatively charged amino acid (aspartic acid or glutamic acid). Stillfurther, it is contemplated that any amino acid residue within the rangeof N1 to N16 can be replaced or substituted. It is envisioned that atleast up to 16 of the N-terminal amino acids residues can be replaced orsubstituted as long as the protein retains it biological and/orfunctional activity, which is stimulating the production of variouscytokines, (e.g., IL-18, MIP-3α, GM-CSF or IFN-γ) by inhibiting variouscytokines, (e.g., IL-2, IL-4, IL-5, IL-10, and TNF-α) by improving acardiovascular disease, e.g., atherosclerosis, or the parametersrelating to cardiovascular disease including circulating levels of totalcholesterol, HDL, LDL, VLDL, trigylcerides and C-reactive protein (CRP).Thus, the N-terminal lactoferrin variants of the present invention areconsidered functional equivalents of lactoferrin.

In terms of functional equivalents, it is well understood by the skilledartisan that, inherent in the definition of a “biologically functionalequivalent” protein is the concept that there is a limit to the numberof changes that may be made within a defined portion of the moleculewhile retaining a molecule with an acceptable level of equivalentbiological activity and/or enhancing the biological activity of thelactoferrin molecule. Biologically functional equivalents are thusdefined herein as those proteins in which selected amino acids (orcodons) may be substituted. Functional activity is defined as theability of lactoferrin to stimulate or inhibit various cytokines orchemokines and/or by improving a cardiovascular disease, e.g.,atherosclerosis, or the parameters relating to cardiovascular diseaseincluding circulating levels of total cholesterol, HDL, LDL, VLDL,trigylcerides and C-reactive protein (CRP).

Still further, the N-terminal amino acid residues can be substitutedwith a modified and/or unusual amino acids. A table of exemplary, butnot limiting, modified and/or unusual amino acids is provided hereinbelow. TABLE 1 Modified and/or Unusual Amino Acids Abbr. Amino AcidAbbr. Amino Acid Aad 2-Aminoadipic acid EtAsn N-Ethylasparagine BAad3-Aminoadipic acid Hyl Hydroxylysine BAla beta-alanine, beta-Amino- AHylallo-Hydroxylysine propionic acid Abu 2-Aminobutyric acid 3Hyp3-Hydroxyproline 4Abu 4-Aminobutyric acid, 4Hyp 4-Hydroxyprolinepiperidinic acid Acp 6-Aminocaproic acid Ide Isodesmosine Ahe2-Aminoheptanoic acid Aile allo-Isoleucine Aib 2-Aminoisobutyric acidMeGly N-Methylglycine, sarcosine BAib 3-Aminoisobutyric acid MeIleN-Methylisoleucine Apm 2-Aminopimelic acid MeLys 6-N-Methyllysine Dbu2,4-Diaminobutyric acid MeVal N-Methylvaline Des Desmosine Nva NorvalineDpm 2,2′-Diaminopimelic acid Nle Norleucine Dpr 2,3-Diaminopropionicacid Orn Ornithine EtGly N-Ethylglycine

The presence and the relative proportion N-terminal lactoferrin variants(deletions and/or substitutions) in a preparation of lactoferrin(lactoferrin composition) may be done by determination of the N-terminalamino acid sequence by the process of Edman degradation using standardmethods. A relative proportion of N-terminal lactoferrin variantcomprise at least 1% of the lactoferrin composition, at least 5% of thelactoferrin composition, at least 10% of the lactoferrin composition, atleast 25% of the lactoferrin composition, at least 50% of thelactoferrin composition or any range in between.

In this method, the protein is reacted with phenylisothiocyanate (PITC),which reacts with the amino acid residue at the amino terminus underbasic conditions to form a phenylthiocarbamyl derivative (PTC-protein).Trifluoroacetic acid then cleaves off the first amino acid as itsanilinothialinone derivative (ATZ-amino acid) and leaves the new aminoterminus for the next degradation cycle.

The percentage of N-terminal lactoferrin variant may also be done moreprecisely by using a Dansylation reaction. Briefly, protein isdansylated using Dansyl chloride reacted with the protein in alkalineconditions (pH 10). Following the Dansylation, the reaction mixtures aredried to pellets, then completely hydrolyzed in 6N HCl. The proportionof N-terminal amino acids are identified by RP HPLC using an in-linefluorometer in comparison with standards made up of known dansylatedamino acids.

B. PHARMACEUTICAL COMPOSITIONS

The present invention is drawn to a composition comprising lactoferrinthat is dispersed in a pharmaceutical carrier, which is used to treatcardiovascular disease. The lactoferrin that is contained in thecomposition of the present invention comprises lactoferrin or anN-terminal lactoferrin variant in which at least the N-1 terminalglycine residue is truncated or substituted. More specifically, theN-terminal lactoferrin variant comprises at least 1% of the composition,at least 5% of the composition, at least 10% of the composition, atleast 25% of the composition, at least 50% of the composition or anyrange in between.

Yet further, the composition comprises lactoferrin in combination with ametal chelator dispersed in a pharmaceutical carrier. Thus, the presentinvention is drawn to a lactoferrin composition with or without a metalchelator that is dispersed in a pharmaceutical carrier. One of skill inthe art understands that both compositions (e.g., lactoferrin alone orlactoferrin in combination with a metal chelator) are within the scopeof the present invention and can be used interchangeably depending uponthe type of response that is desired. It is envisioned that the additionof a metal chelator to the lactoferrin composition enhances thesequestering of metal ions and thus strengthens the immune system orenhances the effect of lactoferrin.

Metal chelators that can be used in combination with lactoferrin,include the divalent metal chelators, for example,ethylenediaminetetraacetic acid (EDTA),[ethylenebis(oxyethylenenitrilo)] tetraacetic acid (EGTA),1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA),hydroxyethlene triamine diacetic acid, (HEDTA) or any salts thereof.More preferably, EDTA is used in combination with lactoferrin.

Further in accordance with the present invention, the composition of thepresent invention suitable for administration is provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, e.g.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent or carrier is detrimental to the recipient or to thetherapeutic effectiveness of a the composition contained therein, itsuse in administrable composition for use in practicing the methods ofthe present invention is appropriate. Examples of carriers or diluentsinclude fats, oils, water, saline solutions, lipids, liposomes, resins,binders, fillers and the like, or combinations thereof.

In accordance with the present invention, the composition is combinedwith the carrier in any convenient and practical manner, e.g., bysolution, suspension, emulsification, admixture, encapsulation,absorption and the like. Such procedures are routine for those skilledin the art.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, e.g.,denaturation in the stomach. Examples of stabilizers for use in an thecomposition include buffers, amino acids such as glycine and lysine,carbohydrates such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc., proteolytic enzymeinhibitors, and the like. Yet further, it is envisioned that divalentmetal chelators, for example EDTA, can also be used to stabilize thecomposition of the present invention. More preferably, for an orallyadministered composition, the stabilizer can also include antagonists tothe secretion of stomach acids.

The composition for oral administration which is combined with asemi-solid or solid carrier can be further formulated into hard or softshell gelatin capsules, tablets, or pills. More preferably, gelatincapsules, tablets, or pills are enterically coated. Enteric coatingsprevent denaturation of the composition in the stomach or upper bowelwhere the pH is acidic. See, e.g., U.S. Pat. No. 5,629,001. Uponreaching the small intestines, the basic pH therein dissolves thecoating and permits the lactoferrin composition to be released andabsorbed by specialized cells, e.g., epithelial enterocytes and Peyer'spatch M cells.

In another embodiment, a powdered composition is combined with a liquidcarrier such as, e.g., water or a saline solution, with or without astabilizing agent.

The compositions of the present invention may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

Sterile injectable solutions are prepared by incorporating thelactoferrin in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Further, the composition for topical administration which is combinedwith a semi-solid carrier can be further formulated into a gel ointment.A preferred carrier for the formation of a gel ointment is a gelpolymer. Preferred polymers that are used to manufacture a gelcomposition of the present invention include, but are not limited tocarbopol, carboxymethyl-cellulose, and pluronic polymers. Gel polymersprevent denaturation of the composition in the open skin by serumproteases.

The amount of lactoferrin in the present invention may vary from about 1ng to about 100 g of lactoferrin, more preferably 1 ng to 20 g per day,most preferably 1 μg to 5 g. In preferred embodiments, the compositionof the present invention comprises a lactoferrin concentration of about0.1% to about 100%. The lactoferrin composition may comprise lactoferrinor an N-terminal lactoferrin variant in which at least the N-1 terminalglycine residue is truncated and/or substituted.

More preferably, the composition of the present invention also containsmetal chelators, for example, but not limited toethylenediaminetetraacetic acid (EDTA), [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA),1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA),hydroxyethlene triamine diacetic acid, (HEDTA) or salts thereof. Theamount of the metal chelator in the composition may vary from about 1 ngto about 1 g. A preferred metal chelator is EDTA.

Upon formulation, solutions are administered in a manner compatible withthe dosage formulation and in such amount as is therapeuticallyeffective to result in an improvement or remediation of the symptoms.The formulations are easily administered in a variety of dosage formssuch as ingestible solutions, drug release capsules and the like. Somevariation in dosage can occur depending on the condition of the subjectbeing treated. The person responsible for administration can, in anyevent, determine the appropriate dose for the individual subject.

C. TREATMENT OR PROPHYLAXIS OF CARDIOVASCULAR DISEASE

In accordance with the present invention, the composition provided inany of the above-described pharmaceutical carriers is administered to asubject who has experienced or is at risk of developing cardiovasculardisease. Risk factors include, but are not limited to elevated levels ofcholesterol or CRP. One of skill in the art can determine the patientswho would potentially benefit from a therapeutic agent that would reducecirculating levels of total cholesterol or triglycerides orcardiovascular inflammation. One of skill in the art can determine thetherapeutically effective amount of the composition to be administeredto a subject based upon several considerations, such as local effects,pharmacodynamics, absorption, metabolism, method of delivery, age,weight, disease severity and response to the therapy.

Oral administration of the composition includes oral, buccal, enteral orintragastric administration. It is also envisioned that the compositionmay be used as a food additive. For example, the composition issprinkled on food or added to a liquid prior to ingestion.

In addition to oral administration, the lactoferrin composition can alsobe administered parenterally, which includes, but is not limited tointradermal, subcutaneous, intramuscular, intraperitoneal, intravenous,intraarterial, intramyocardial, transendocardial, transepicardial,intrathecal, and infusion techniques.

Cardiovascular diseases and/or disorders include, but are not limitedto, diseases and/or disorders of the pericardium, heart valves (e.g.,incompetent valves, stenosed valves, Rheumatic heart disease, mitralvalve prolapse, aortic regurgitation), myocardium (e.g., coronary arterydisease, myocardial infarction, heart failure, ischemic heart disease,angina) blood vessels (e.g., hypertension, arteriosclerosis, aneurysm)or veins (e.g., varicose veins, hemorrhoids). In specific embodiments,the cardiovascular disease is atherosclerosis.

In specific embodiments of the present invention, the lactoferrincomposition is administered to a subject suffering from or at risk fordeveloping atherosclerosis. Thus, it is envisioned that the lactoferrincomposition modulates or reduces the severity and/or incidence ofatherosclerosis.

Prophylactic treatment can be administered to those subjects at risk fordeveloping atherosclerosis. One risk factor is an atherogeniclipoprotein profile. For example, a ratio of serum cholesterol to highdensity lipoproteins of above 5:1 indicates a higher than average riskof developing atherosclerosis. Other factors indicating increased riskfor atherosclerosis include a serum cholesterol level of above 240mg/dl; a high density lipoprotein level below about 35 mg/dl; and a lowdensity lipoprotein level above about 160 mg/dl.

Another embodiment includes treating a human subject with an elevatedlevel of circulating total cholesterol or CRP according to the thenmedically established guidelines. It is contemplated that thelactoferrin composition of the present invention reduces or attenuatesthe levels of circulating total cholesterol, low density lipoproteins orvery low density lipoproteins. It is contemplated that the lactoferrincomposition of the present invention can interfere with how cholesterolenters the circulation either via absorption from food (exogenouspathway) or synthesis by the liver (endogenous pathway).

In further embodiments, the composition is administered in conjunctionwith an antacid. Thus, an antacid is administered prior or substantiallysimultaneously with or after oral administration of the composition. Theadministration of an antacid just prior or immediately following theadministration of the composition may help to reduce the degree ofinactivation of the lactoferrin in the digestive tract. Examples ofappropriate antacids include, but are not limited to, sodiumbicarbonate, magnesium oxide, magnesium hydroxide, calcium carbonate,magnesium trisilicate, magnesium carbonate and alumin hydroxide gel.

In a preferred embodiment of the present invention, the composition isadministered in an effective amount to decrease, reduce, inhibit orabrogate cardiovascular disease. Thus, a subject is administered atherapeutically effective amount of a lactoferrin composition so thatthe subject has an improvement in the parameters relating tocardiovascular disease including circulating levels of totalcholesterol, HDL, LDL, VLDL, trigylcerides and C-reactive protein (CRP).The amount of lactoferrin in the composition may vary from about 1 ng toabout 20 g. Preferably, the composition that is orally administeredcontains the range of 0.5 g to 5 g of lactoferrin per day.

The improvement is any observable or measurable improvement. Thus, oneof skill in the art realizes that a treatment may improve the patientcondition, but may not be a complete cure of the disease. In certainaspects, the composition is administered in an effective amount todecrease, reduce, inhibit or abrogate excess amounts of cholesterollevels in circulation. A subject requires treatment for cholesterollevels based upon any of the following situations: LDL of 160 mg/ml orgreater; LDL of 130-159 mg/ml and also have two or more cardiovascularrisk factors; LDL of 100 mg/ml or greater in subjects with coronaryheart disease (CHD); triglycerides of 200 mg/dl or higher; totalcholesterol of 240 mg/dl or higher or HDL of less than 40 mg/dl. Thus,after administration of lactoferrin, if any of the above conditionsimprove, then the amount of lactoferrin is considered an effectiveamount to decrease, reduce, inhibit or abrogate cholesterol levels inthe circulation.

Another embodiment is a method of reducing vascular inflammation byadministering the lactoferrin composition of the present invention.Vascular inflammation can be tied to a number of the underlyingprocesses contributing to atherosclerosis which include endothelialdysfunction, vascular proliferation and matrix alteration. Recentstudies have emphasized the involvement of inflammation in mediating allstages of atherosclerosis. Vascular inflammation is thought to be aconsequence of damage to the vascular endothelium and may also involvethe proliferation of vascular smooth muscle cells (vsmcs). One precursorof lesion development in humans may be focal accumulation of vsmcswithin the intima. In early atherosclerosis, vsmcs may contribute to thedevelopment of the atheroma through the production of pro-inflammatorymediators such as monocyte chemoattractant protein 1 and vascular celladhesion molecule, and through the synthesis of matrix moleculesrequired for the retention of lipoproteins. Inflammation of the vascularendothelium and proliferation of vsmcs may also impact the stability ofthe plaque through the formation of a firm fibrous cap. Indeed, inlipid-laden lesions in which the fibrous cap is thin and weak, there isevidence of vsmc apoptosis, especially at the “shoulder” region,associated with inflammation. In addition, the local inflammatory milieucan induce expression of collagenase and inhibit expression ofproteolytic inhibitors, thus rendering the fibrous cap weak andsusceptible to rupture. Lactoferrin, having known anti-inflammatoryproperties, may thus serve to inhibit the underlying processesassociated with the development of atherosclerosis.

In further embodiments, the lactoferrin composition may also reducevascular spasms or vascular hyper-reactivity. Vascular spasms are asudden, brief tightening of a blood vessel, which can temporarily reduceblood flow to tissues supplied by that vessel.

Still further, the lactoferrin composition may also promote endothelialintegrity or healing. Endothelia are the layer of cells lining the bloodvessels. Endothelial dysfunction most commonly refers to impairment ofendothelium-dependent vasodilation and widespread abnormalities inendothelial integrity and homeostasis. It is believed that HDLs helpmaintain endothelial integrity, facilitate vascular relaxation, inhibitblood cell adhesion to vascular endothelium, reduce plateletaggregability and coagulation, and may favor fibrinolysis. The integrityor completeness of the endothelia lining of the vessels is important topreventing/treating the development of plaques and atherosclerosis.Thus, it is envisioned that the lactoferrin composition of the presentinvention will promote or modulate endothelial integrity or healing.

Yet further, another embodiment is a method of preventing acardiovascular disease in a subject at risk for developing acardiovascular disease comprising the step of administering to thesubject a lactoferrin composition in an amount sufficient to result inprophylaxis of the cardiovascular disease in the subject. In preferredembodiments, the cardiovascular disease is atherosclerosis. It isenvisioned that the lactoferrin composition not only possess therapeuticbenefits for those subjects suffering from cardiovascular diseases, butalso possess prophylactic properties for those subjects at risk fordeveloping cardiovascular disease. A subject at risk may or may not becognizant of their disease state or potential disease state and may ormay not be aware that they are need of treatment.

Thus, prophylatically, it is envisioned that the lactoferrin compositioncan reduce any of the following: the levels of circulating totalcholesterol, low density lipoproteins (LDL), very low densitylipoproteins (VLDL), levels of vascular inflammation, circulatingC-reactive protein (CRP), triglycerides, and the proliferation ofvascular smooth muscle cells in the subject. Yet further, thelactoferrin composition may also increase the levels of circulating highdensity lipoproteins (HDL).

Still yet, a further embodiment is a method of enhancing immune responsein a subject comprising the step of administering to the subject thecomposition of the present invention. It is envisioned that the immuneresponse, whether local, systemic or mucosal, is enhanced by lactoferrinstimulating cytokines and/or chemokines. Exemplary cytokines includeinterleukin-18 and GM-CSF in the gastrointestinal tract, which are knownto enhance immune cells or stimulate production of immune cells. Forexample, interleukin-18 enhances natural killer cells or T lymphocytes.In specific embodiments, interleukin-18 (IL-18) enhances CD4+, CD8+ andCD3+cells. It is known by those of skill in the art that IL-18 is a Th₁cytokine that acts in synergy with interleukin-12 and interleukin-2 inthe stimulation of lymphocyte IFN-gamma production. Other cytokines orchemokines may also be enhanced for example, but not limited to IL-12,IL-1b, MIP-3α, MIP-1α or IFN-γ. Other cytokines or enzymes may beinhibited for example, but not limited to IL-2, IL-4, IL-5, IL-10,TNF-α, or matrix metalloproteinases. It is further contemplated thatlactoferrin inhibits the production of TNF-α, which inhibits cellsinvolved in inflammation. It is also envisioned that lactoferrinstimulates interleukin-18 and a Th₁ response following oraladministration, which inhibits pro-inflammatory cytokines, e.g., IL-4,IL-5, IL-6, IL-8 and TNF-α.

The lactoferrin composition of the present invention can also result ininhibition of a cytokine or chemokine. The cytokines include, but arenot limited to interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5(IL-5), interleukin-10 (IL-10), and tumor necrosis factor alpha (TNF-α).Still further, the lactoferrin composition can also inhibit theproduction of matrix metalloproteinases (MMPs).

In further embodiments, cytokines, for example, interleukin-18 orgranulocyte/macrophage colony-stimulating factor, can stimulate theproduction or activity of immune cells. The immune cells include, butare not limited to T lymphocytes, natural killer cells, NK-T cells,macrophages, dendritic cells, and polymorphonuclear cells. Morespecifically, the polymorphonuclear cells are neutrophils and the Tlymphocytes are selected from the group consisting of CD4+, CD8+ andCD3+T cells.

In further embodiments, the composition of the present invention alsocontains metal chelators, for example, but not limited toethylenediaminetetraacetic acid (EDTA), [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA),1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA),hydroxyethlene triamine diacetic acid, (HEDTA) or salts thereof.

Treatment regimens may vary as well, and often depend on the health andage of the patient. Obviously, certain types of disease will requiremore aggressive treatment, while at the same time, certain patientscannot tolerate more taxing regimens. The clinician will be best suitedto make such decisions based on the known efficacy and toxicity (if any)of the therapeutic formulations.

In specific embodiments, the composition is given in a single dose ormultiple doses. The single dose may be administered daily, or multipletimes a day, or multiple times a week, or monthly or multiple times amonth. In a further embodiment, the lactoferrin is given in a series ofdoses. The series of doses may be administered daily, or multiple timesa day, weekly, or multiple times a week, or monthly, or multiple times amonth.

D. COMBINATION TREATMENTS

In order to increase the effectiveness of the composition, it may bedesirable to combine these compositions and methods of the inventionwith a known agent effective in the treatment or prevention ofcardiovascular disease or disorder, for example known agents to treat orprevent atherosclerosis. In some embodiments, it is contemplated that aconventional therapy or agent, including but not limited to, apharmacological therapeutic agent, a surgical therapeutic agent (e.g., asurgical procedure) or a combination thereof, may be combined with thecomposition of the present invention.

The composition of the present invention may precede, be co-current withand/or follow the other agent(s) by intervals ranging from minutes toweeks. In embodiments where the composition of the present invention,and other agent(s) are applied separately to a cell, tissue or organism,one would generally ensure that a significant period of time did notexpire between the time of each delivery, such that the composition andagent(s) would still be able to exert an advantageously combined effecton the cell, tissue or organism.

Various combination regimens of the composition and one or more agentsare employed. One of skill in the art is aware that the composition ofthe present invention and agents can be administered in any order orcombination. In other aspects, one or more agents may be administeredsubstantially simultaneously, or within about minutes to hours to daysto weeks and any range derivable therein, prior to and/or afteradministering the composition.

Administration of the composition to a cell, tissue or organism mayfollow general protocols for the administration of cardiovasculartherapeutics, taking into account the toxicity, if any. It is expectedthat the treatment cycles would be repeated as necessary. In particularembodiments, it is contemplated that various additional agents may beapplied in any combination with the present invention.

A. Pharmacological Therapeutic Agents

Pharmacological therapeutic agents and methods of administration,dosages, etc. are well known to those of skill in the art (see forexample, the “Physicians Desk Reference”, Goodman & Gilman's “ThePharmacological Basis of Therapeutics”, “Remington's PharmaceuticalSciences”, and “The Merck Index, Eleventh Edition”, incorporated hereinby reference in relevant parts), and may be combined with the inventionin light of the disclosures herein. Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject, and suchindividual determinations are within the skill of those of ordinaryskill in the art.

Non-limiting examples of a pharmacological therapeutic agent that may beused in the present invention include an antihyperlipoproteinemic agent,an antiarteriosclerotic agent, an anti-cholesterol agent, ananti-inflammatory agent, an antithrombotic/fibrinolytic agent, a bloodcoagulant, an antiarrhythmic agent, an antihypertensive agent, or avasopressor. In certain aspects of the present invention,anti-cholesterolemic agents are used in combination with the compositionof the present invention. Anti-cholesterol agents include but are notlimited to HMG-CoA Reductase inhibitors, cholesterol absorptioninhibitors, bile acid sequestrants, nicotinic acid and derivativesthereof, fibric acid and derivatives thereof. HMG-CoA Reductaseinhibitors include statins, for example, but not limited to atorvastatincalcium (Lipitor®), cerivastatin sodium (Baycol®), fluvastatin sodium(Lescol®), lovastatin (Advicor®), pravastatin sodium (Pravachol®), andsimvastatin (Zocor®). Agents known to reduce the absorption of ingestedcholesterol include, for example, ezetimibe (Zetia®). Bile acidsequestrants include, but are not limited to cholestryramine,cholestipol and colesevalam. Other anti-cholesterol agents includefibric acids and derivatives thereof (e.g., gemfibrozil, fenofibrate andclofibrate); nicotinic acids and derivatives thereof (e.g., nician,lovastatin) and agents that extend the release of nicotinic acid, forexample niaspan. Anti-inflammatory agents include, but are not limitedto non-sterodial anti-inflammatory agents (e.g., naproxen, ibuprofen,celeoxib) and sterodial anti-inflammatory agents (e.g.,glucocorticoids).

B. Surgical Therapeutic Agents

In certain aspects, a therapeutic agent may comprise a surgery of sometype, which includes, for example, preventative, diagnostic or staging,curative and palliative surgery. Surgery, and in particular a curativesurgery, may be used in conjunction with other therapies, such as thepresent invention and one or more other agents.

Such surgical therapeutic agents for cardiovascular diseases anddisorders are well known to those of skill in the art, and may comprise,but are not limited to, performing surgery on an organism, providing acardiovascular mechanical prostheses, angioplasty, coronary arteryreperfusion, catheter ablation, providing an implantable cardioverterdefibrillator to the subject, mechanical circulatory support or acombination thereof. Non-limiting examples of a mechanical circulatorysupport that may be used in the present invention comprise anintra-aortic balloon counterpulsation, left ventricular assist device orcombination thereof.

E. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Cholesterol Reduction by Recombinant Human Lactoferrin (rhLF)

Healthy adult human volunteers were administered a liquid formulation ofeither rhLF or placebo for seven days. Fasting serum was collected atbaseline (prior to administration of rhLF) and at the end of study (Day10) and levels of total cholesterol determined. All subjects were housedin an in-patient setting for the duration of the study and receivedsimilar diets.

In this clinical trial, human subjects treated with placebo drugexperienced a rise in serum cholesterol. RhLF treated subjects in asimilar setting and receiving a similar diet, experienced a reduction intotal cholesterol. FIG. 1 shows that administration of rhLF for justseven days resulted in 14% reduction in total cholesterol (P<0.05).

Example 2 Reduction of C-Reactive Protein (CRP) by Recombinant HumanLactoferrin (rhLF)

Healthy adult human volunteers were administered a liquid formulation ofrhLF or placebo for seven days. RhLF was administered for a total ofseven days. Serum was collected on Day 1 and Day 7 of rhLFadministration and assayed for CRP using a high sensitive assay.

A total of six subjects had Day 1 CRP levels that were measurable by thehigh sensitive assay (>0.07 mg/dL). Five out of the six subjects showeda reduction in CRP with the sixth patient showing no change. As shown inFIG. 2, the six evaluable subjects showed an average of 49% reduction inCRP levels (P<0.05) as well as a 17% reduction in their cardiovascularrisk.

Example 3 RhLF Effect on Hyperlipidemia in Mice

The effect of oral rhLF was tested in a mouse model of hyperlipidemiainduced by the mice being fed a high cholesterol-fat diet for 14 days (2g lard, 8 g coconut oil, 1 g cholesterol, 0.3 g cholic acid per 100 g offeed and 88.7 g standard chow). The hyperlipidemic mice wereadministered either placebo vehicle or rhLF (1000 mg/kg) twice a day forseven days. Twenty-four hours after the last dose, serum was obtainedfrom individual fasting animals and assayed for total cholesterol, HDLand LDL. The rhLF treated mice showed a trend toward decrease in totalcholesterol (16%) and LDL cholesterol (23%) and an increase in HDLcholesterol (15%). The HDL/LDL ratio was increased by 48.5% and wasstatistically significant (FIG. 3).

Example 4 Dose Effects of RhLF on Hyperlipidemia in Mice

Mice were rendered hypercholesteremic by administration of a highcholesterol diet for fourteen days. Animals received rhLF (32.5, 150 or500 mg/kg) or placebo administered orally twice a day for 14 consecutivedays. Twenty-four hours after the last dose of drug, fasting animalswere sacrificed and serum total cholesterol (Total, high densitylipoprotein (HDL), low density lipoprotein (LDL) and Total/HDL) ratiowere determined.

High cholesterol diet used to induce hyperlipidemia consisted of 2 glard, 8 g coconut oil, 1 g cholesterol, 0.3 g cholic acid per 100 g offeed and 88.7 g standard chow.

At the doses tested, a beneficial effect from rhLF was observed after 14days treatment. No dose dependence was observed. As shown in FIG. 4, alldoses of rhLF decreased both total cholesterol and LDL-cholesterolrelataive to placebo treated animals. A statistically significantdecrease in LDL-cholesterol was observed (25% decrease, p<0.05) when allthe rhLF treated animals were compared to the placebo treated animals.There was also a decrease in total cholesterol (19% reduction;p=0.0594).

Example 5 Effect of Oral rhLF with Lovastatin on Hyperlipidemia in Mice

The ability of oral rhLF to potentiate the effect of lovastatin in theinduced hyperlipidemia model was also tested. Hyperlipidemic mice weretreated with lovastatin (15 mg/kg) alone or in combination with rhLF(500 mg/kg b.i.d.) for seven days. Mice treated with lovastatin pluslactoferrin had astatistically significant (p<0.05) reduction in bothTotal Cholesterol and LDL-Cholesterol relative to the lovastatin treatedmice (18% and 25% respectively) and a 13% increase in HDL cholesterol(FIG. 5).

Example 6 Reduction of Systemic Inflammation in Murine Models

Mice and rats with elevated systemic or cardiovascular inflammation areadministered placebo or rhLF for 7, 14 or 28 days and markers ofinflammation including CRP are assayed.

Example 7 Reduction of Cholesterol with rhLF therapy

Human patients with elevated levels of cholesterol (>200 mg/dL) areadministered rhLF or placebo for 14, 28 and 90 days. Fasting serum isassayed for total cholesterol, HDL, LDL, VLDL, and triglycerides.

Example 8 Reduction of CRP with rhLF therapy

Human patients with elevated levels of CRP are administered rhLF orplacebo for 14, 28 and 90 days. Serum is assayed for CRP and othermarkers of inflammation.

Example 9 Dose Ranging Study of rhLF in the Reduction of Cholesterol andCRP

Human patients with elevated levels of cholesterol (>200 mg/dL) aregiven placebo or ascending doses of rhLF for 30, 90 and 180 days.Fasting serum is assayed for total cholesterol, HDL, LDL, VLDL,triglycerides and CRP.

Example 10 Reduction of Cholesterol and CRP with rhLF in CombinationTherapy

Human patients with elevated levels of cholesterol (>200 mg/dL) areadministered an approved cholesterol reducing drug either alone or incombination with rhLF for 30, 90 and 180 days. Cholesterol reducingdrugs tested include Lipitor® (atorvastatin), Zocor® (simvastatin), andZetia® (ezetimibe). Fasting serum is assayed for total cholesterol, HDL,LDL, VLDL, triglycerides and CRP.

Example 11 Reduction of Cardiovascular Incidence with rhLF Alone or inCombination Therapy

Human patients considered at an elevated risk for cardiovascularaccidents (including stroke and heart attacks) are treated with rhLFalone, approved drugs alone or a combination of rhLF and an approveddrug. Fasting serum is assayed for total cholesterol, HDL, LDL, VLDL,triglycerides and CRP. Incidence and severity of stroke and heartattacks and incidence of mortality are also measured.

REFERENCES CITED

All patents and publications mentioned in the specifications areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

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Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the invention asdefined by the appended claims. Moreover, the scope of the presentapplication is not intended to be limited to the particular embodimentsof the process, machine, manufacture, composition of matter, means,methods and steps described in the specification. As one will readilyappreciate from the disclosure, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized. Accordingly, the appended claims areintended to include within their scope such processes, machines,manufacture, compositions of matter, means, methods, or steps.

1-31. (canceled)
 32. A method of modulating atherosclerosis in a subjectcomprising the step of administering to said subject an effective amountof a lactoferrin composition to modulate atherosclerosis in saidsubject.
 33. The method of claim 32, wherein modulating is reducing theincidence of atherosclerosis in said subject.
 34. The method of claim32, wherein modulating is reducing the severity of atherosclerosis insaid subject.
 35. The method of claim 32, wherein said lactoferrincomposition reduces levels of circulating total cholesterol, low densitylipoproteins (LDL), very low density lipoproteins (VLDL), ortriglycerides in said subject.
 36. The method of claim 32, wherein saidlactoferrin composition increases the levels of circulating high densitylipoproteins (HDL) in said subject.
 37. The method of claim 32, whereinsaid lactoferrin composition reduces the levels of vascular inflammationin said subject.
 38. The method of claim 32, wherein said lactoferrincomposition reduces circulating C-reactive protein (CRP) in saidsubject.
 39. The method of claim 32, wherein said lactoferrincomposition reduces the proliferation of vascular smooth muscle cells insaid subject.
 40. The method of claim 32, wherein said lactoferrincomposition reduces the vascular spasm or vascular hyper-reactivity insaid subject.
 41. The method of claim 32, wherein said lactoferrincomposition promotes endothelial integrity or healing in said subject.42. The method of claim 32, wherein said lactoferrin composition isdispersed in a pharmaceutically acceptable carrier.
 43. The method ofclaim 32, wherein said lactoferrin is mammalian lactoferrin.
 44. Themethod of claim 43, wherein said lactoferrin is human or bovine.
 45. Themethod of claim 32, wherein said lactoferrin is recombinant lactoferrin.46. The method of claim 32, wherein said lactoferrin compositioncomprises an N-terminal lactoferrin variant.
 47. The method of claim 46,wherein the N-terminal lactoferrin variant lacks at least the N-terminalglycine residue.
 48. The method of claim 47, wherein said N-terminallactoferrin variant comprises at least 1% to at least 50% of thelactoferrin composition.
 49. The method of claim 32, wherein saidlactoferrin composition is administered parenterally.
 50. The method ofclaim 49, wherein parenterally is subcutaneously, intramuscularly,intraperitoneally, intravenously, intraarterially, intramyocardially,transendocardially, transepicardially, or intrathecally.
 51. The methodof claim 32, wherein said lactoferrin composition is administeredorally.
 52. The method of claim 51 further comprising administering anantacid in conjunction with said lactoferrin composition.
 53. The methodof claim 51 further comprising administering the lactoferrin in adelayed release formulation.
 54. The method of claim 53 where thelactoferrin release occurs in the small intestine.
 55. The method ofclaim 53 where the lactoferrin release occurs in the large intestine.56. The method of claim 32, wherein the amount of the lactoferrin thatis administered is about 1 ng to about 20 g per day.
 57. The method ofclaim 32, wherein the amount of the lactoferrin that is administered isabout 0.1 g to about 5 g per day.
 58. The method of claim 32, whereinsaid lactoferrin composition reduces the production or activity ofpro-inflammatory cytokines.
 59. The method of claim 32 furthercomprising administering a lactoferrin composition in combination withan anti-cholesterol agent or an anti-inflammatory agent.
 60. The methodof claim 59, wherein the anti-cholesterol agent is selected from thegroup consisting of cholesterol absorption inhibitors, bile acidsequestrants, nicotinic acid, fibric acids and HMG-coA reductaseinhibitors.
 61. The method of claim 60, wherein the bile acidsequestrants are selected from the group consisting of cholestryramine,cholestipol and colesevalam.
 62. The method of claim 60, wherein thefibric acids are selected from the group consisting of gemfibrozil,fenofibrate and clofibrate.
 63. The method of claim 60, wherein theHMG-coA reductase inhibitors are selected from the group consisting oflovastatin, pravastatin, simvastatin, fluvastatin, atorvastatin andcerivastatin.